Electrophoresis is an analytic technique used in various biochemical analyses relating to nucleic acids such as DNA and RNA, amino acids and proteins and the like. Recently devices have been proposed that are capable of analyzing fluorescence emitted from a small amount of a solution sample in the order of nanoliters to picoliters by labeling the solution sample with an appropriate fluorophore when the sample is irradiated in a compact electrophoresis chip with an excitation light. The present inventors have also provided to date such devices and methods, which are disclosed in Patent document 1 and Non-Patent literature 1 by listed below.
FIGS. 4 (A) and (B) illustrate an example of a previous device disclosed by the present inventors in the Patent document 1 and Non-Patent literature 1. With reference to the drawings, the device has a chip 10 for containing and supporting an analytical sample. The chip 10 is provided with mutually planar intersecting microchannels 15 and 16. One of these, channel 15 called an injection channel, is provided at one end thereof with a well-shaped sample reservoir 11 for containing a sample in the form of a solution, and at the other end thereof with a waste reservoir 12 for receiving the sample flowing out via the injection channel 15. The other channel 16 which intersects the injection channel 15, called a separation channel, is provided at one end thereof with a cathode reservoir 13 and at the other end thereof with an anode reservoir 14. The reservoirs 11 to 14 are each provided with electrodes (not shown) in the form such as a thin-film electrode, for example, or inserted electrodes that are needle-shaped or the like, for the purpose of applying individually preset voltages at the timings described below. The channels 15 and 16 generally intersect each other orthogonally, as illustrated, forming the shape of a cross in a plan view.
When a sample is loaded into the sample reservoir 11 and an appropriate voltage is applied between the sample reservoir 11 and the waste reservoir 12, the sample migrates into the injection channel 15. At this time, the cathode reservoir 13 and the anode reservoir 14 are kept in a floating potential state or an appropriate bias voltage is applied therebetween. When the voltage is switched after the lapse of an appropriate time (generally in the order of 10 to 60 seconds) and an appropriate voltage is applied between the cathode reservoir 13 and the anode reservoir 14, a portion of the sample (called the sample plug) that has just reached the point of intersection with the separation channel 16 is excised, and electrophoresis begins inside the separation channel 16. Further, at this time, an appropriate bias voltage is applied between the sample reservoir 11 and the waste reservoir 12 so that the residual sample that remains in the injection channel 15 does not flow into the separation channel 16.
By utilizing recent semiconductor microfabrication technology, it is possible to form the channels 15 and 16 to a very fine width with good accuracy, and therefore form a short sample plug corresponding to the channel width (generally some tens of micrometers). In practice, the chip 10 is usually manufactured by bonding two glass sheets together, since it is required to have the highest possible optical transmittance in the wavelength of at least the excitation light or fluorescence and to have a good insulating property to the electrophoresis. The channels 15 and 16 are lithographically (in some cases, mechanically) formed on one glass sheet 10a, and subsequently thermal bonding is used to affix the other glass sheet 10b which occludes the channels 15 and 16 from above and which is perforated with vertical holes to form the reservoirs 11-14. It is also possible to use plastic substrates. The two plate members are bonded together by using thermal bonding, ultrasonic welding, or an adhesive agent. It should be noted that this invention does not impose any particular restriction on the structure of this part. It only needs to have a structure suitable for analysis; a conventional configuration may of course be used.
Thus, even with existing fabrication technology, an extremely short sample plug can be obtained, so electrophoretic separation with a high number of theoretical plates can be achieved by using a microfluidic electrophoresis chip having a short channel length. As mentioned above, the sample migrating inside the separation channel 16 is labeled in advance with an appropriate fluorophore. When it is irradiated with an excitation light Le, it therefore emits a light of a different wavelength from the excitation light, generally fluorescence. When the labeled sample plug is migrating inside the separation channel 16, the sample plug reaches to the detection region Po while being separated according to differences in the size of the sample plug and the electric charge and so forth. A so-called electropherogram (electrophoresis data) can be obtained by plotting the intensity of the fluorescence emitted as a result of being irradiated in the detection region Po with the excitation light Le against the time required for the sample plug to reach the detection region Po.
FIG. 4 (B) shows a conventional-construction fluorescence detecting module 40 for detecting the fluorescence. The fluorescence detecting module 40 has a semiconductor light detecting element 20, which in the illustrated cross section appears to be a lateral pair of elements. In fact, as can be seen in a plan view, it is a doughnut-shaped element with a center pinhole 41 through which the excitation light Le passes to irradiate the sample. When this excitation light Le impinges on the chip 10 transparent to the light and irradiates the sample in the separation channel 16 inside the detection region Po, the sample emits fluorescence Lf. Then, the fluorescence Lf is transformed by a microlens 61 for collecting the fluorescence preferably into nearly parallel rays and enters an optical filter 50 disposed on the incidence plane side of the semiconductor light detecting element 20. The optical filter 50 is generally configured as an optical interference filter formed by coating one surface side of a quartz glass 52, and is able to selectively transmit the fluorescence Lf in order to remove as much of the scattered excitation light Le as possible and allow just the fluorescence Lf to fall incident onto the semiconductor light detecting element 20. The fluorescence collecting microlens 61 may be formed integrally with the chip 10 by cast molding or may be formed on a special base plate 61′, as partially depicted in FIG. 4 (B) by an imaginary line, and bonded to the rear surface of the chip 10.
The specific structure of the optical interference filter 50 or the semiconductor light detecting element 20 may be an existing structure, as described below with reference to the embodiments of the invention. That is, modifying the basic structure of the members 50 and 20 is not an object of the invention. As described in the Patent Document 1 and Non-patent Document 1, the semiconductor light detecting element 20 used for the detecting device of the invention preferably comprises a photodiode fabricated using hydrogenated amorphous silicon (a-Si:H).
That is because a-Si:H photodiode has various desirable characteristics, not only in the case of the electrophoresis method but also in the case of biochemical analysis, as listed below.
1) A fluorescence band of fluorophores (such as, for example, Fluorescein, Green Fluorescence Protein, TOTO, and Ethidium Bromide) is located in a visible light region, in which a-Si:H has a high absorption coefficient with respect to visible light.
2) A dark current of a-Si:H is several orders of magnitude lower than that of crystalline silicon, so an a-Si:H photodiode does not require cooling, which is advantageous with respect to decreasing device size.
3) A-Si:H can be patterned by using semiconductor microfabrication technology, facilitating the fabrication of photodiode detector arrays.
4) A-Si:H photodiodes have good mass-producibility and can be formed directly on cheap glass or plastic substrates by using plasma enhanced chemical vapor deposition, facilitating low-cost implementation.
The present inventors actually fabricated an integrated a-Si:H photodiode as disclosed in the Non-document 1 mentioned and tested it using an argon ion laser (excitation wavelength λ=488 nm) as an excitation light source. When the fabricated photodiode was evaluated by detecting fluorescence from a fluorescein fluorophore, the limit of detection was found to be 17 nanomole/L (hereinafter abbreviated as “nM”) for fluorescein concentration. As disclosed in Non-Patent literature 2 listed below, the inventors also fabricated a fluorescence detecting element in which the optical interference filter was integrated monolithically on the a-Si:H photodiode, and tested the element using a solid-state laser (excitation wavelength λ=488 nm) as the excitation light source. The result showed that the detection limit was further decreased, to a fluorescein concentration of 7 nM. Based on those results, the device showed the highest detection sensitivity among the several examples of fluorescence detector of this sort reported to date. This device was in fact successfully applied to the analysis of microfluidic DNA fragments and enantiomers of amino acids. As further variations, Patent document 2, listed below, discloses using a micro-resonator type light-emitting diode (LED) as an excitation light source, and adapting as an excitation light source an LED having an emission aperture no wider than a micro-object.
The final target to be attained by these fluorescence detecting devices is the realization of a so-called lab-on-a-chip or micro total analysis system (μTAS). That is, the target is to integrate and miniaturize, on a single chip, all the elements necessary for a series of analytic processes or analysis, enabling “point-of-care” analysis. To some extent the concepts of the method of fluorescence detection analysis were indeed established prior to the disclosures of Patent documents 1 and 2 and Non-Patents literature 1 and 2. Actually, in the case of microfluidic electrophoresis, high-speed genotyping using 96 to 384 channels was in fact carried out. Moreover, microfluidic valves and pumps were proposed and made available for enabling large-scale parallel microfluidic operations, making it possible to perform microfluidic cell sorting and combinatorial optimization for protein crystallization conditions in large-scale integrated microchambers.
Analytical processes such as electrophoresis and sample preparation processes have become successfully integrated and miniaturized and enabled partially to undergo large-scale integration. However, in most cases, a laser-induced fluorescence detection system composed of a photomultiplier, a CCD, an optical interference filter, and a laser is used for microfluidic lab-on-a-chip analysis, due to the need for high-sensitivity detection of microsamples. Such a system can hardly be called a readily portable device suitable for “point-of-care” analysis. In this respect, the aforementioned system proposed by the present inventors has built a foundation for realizing “point-of-care” biochemical analysis with high speed and low sample consumption. When this success is further developed to the point of the construction and practical realization of a lab-on-a-chip, the lab-on-a-chip will be useful enough for the prompt detection and identification of pathogens scattered by so-called bioterrorism, diagnosing genetic diseases, and performing stress monitoring and the like, and therefore can be expected to have a huge industrial impact.
Patent document 1: JP2005-535871 (B)
Patent document 2: JP2008-039655 (A)
Non-Patent literature 1: T. Kamei et al., “Integrated Hydrogenated Amorphous Si Photodiode Detector for Microfluidic Bioanalytical Devices,” Anal. Chem., Vol. 75, No. 20(Oct. 15, 2003), pp.5300-5305.
Non-Patent literature 2: T. Kamei et al., “Contact-lens type of micromachined hydrogenated amorphous Si fluorescence detector coupled with microfluidic electrophoresis devices”, Appl. Phys. Lett., Vol. 89, pp. 114101-1-3 (2006)